Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn’s disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own.
In this study, we aimed to investigate the association of six well-characterized polymorphisms in tumor necrosis factor alpha and beta (TNF-α and TNF-β) genes with the risk for sarcoidosis via a comprehensive meta-analysis.
Methods And Findings
The electronic MEDLINE (Ovid) and PubMed databases covering the period from the earliest possible year to June 2013 were searched. Total 13 qualified articles including 1584 patients with sarcoidosis and 2636 controls were recruited. The data were analyzed by RevMan software, and risk estimates were expressed as odds ratios (ORs) and 95% confidence intervals (95% CIs). Analyses of the full data set failed to identify any significant association of TNF-α gene -307A (OR=1.25; 95% CI: 0.98-1.59), -1031C (OR=0.88; 95% CI: 0.71-1.1), -863A (OR=0.89; 95% CI: 0.72-1.11), -238A (OR=0.97; 95% CI: 0.71-1.32), and -857T (OR=1.14; 95% CI: 0.74-1.77) alleles, but a significant association for TNF-β 252A allele (OR=1.65; 95%CI = 1.33-2.04; P<0.00001). Under a random-effects allelic model, there was marginally significant increased risk of sarcoidosis for -307A allele among Caucasians (OR=1.25; 95% CI: 0.96-1.62; P=0.09) but not among Asians (OR=2.12; 95% CI: 0.31-14.27; P=0.44). There was a low probability of publication bias as reflected by the fail-safe number.
This meta-analysis extended previous findings on the association between the TNF-α and TNF-β genetic polymorphisms and sarcoidosis, by showing that the TNF-β gene A252G polymorphism might be a potential risk factor for the development of sarcoidosis.
Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that, for 2-Arachidonoylglycerol (2-AG), is mediated by monoacylglycerol lipase (MAGL). The piperidine carbamate, 4-nitrophenyl- 4-(dibenzo[d] [1,3]dioxol-5-yl (hydroxy) methyl) piperidine- 1-carboxylate (JZL184), is a drug that inhibits MAGL and presents high potency and selectivity. Thus, JZL184 increases the levels of 2-AG, an endocannabinoid that acts on the CB1 and CB2 cannabinoid receptors. Here, we investigated the effects of MAGL inhibition, with a single dose (16 mg/kg, intraperitoneally (i.p.)) of JZL184, in a murine model of lipopolysaccharide (LPS) -induced acute lung injury (ALI) 6, 24 and 48 hours after the inflammatory insult. Treatment with JZL184 decreased the leukocyte migration into the lungs as well as the vascular permeability measured through the bronchoalveolar lavage fluid (BAL) and histological analysis. JZL184 also reduced the cytokine and chemokine levels in the BAL and adhesion molecule expression in the blood and BAL. The CB1 and CB2 receptors were considered involved in the anti-inflammatory effects of JZL184 because the AM281 selective CB1 receptor antagonist (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and the AM630 selective CB2 receptor antagonist ([6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone) blocked the anti-inflammatory effects previously described for JZL184. It was concluded that MAGL inhibition, and consequently the increase in 2-AG levels, produced anti-inflammatory effects in a murine model of LPS-induced ALI, a finding that was considered a consequence of the activation of the CB1 and CB2 receptors.
A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-α and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-κB subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-α mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-α-induced inflammatory signaling and NF-κB-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.
Behavioral inhibition reflects a disposition to react warily to novel situations, and has been associated with atopic diseases such as asthma. Retrospective work established the relationship between behavioral inhibition in rhesus monkeys (Macaca mulatta) and airway hyperresponsiveness, but not atopy, and the suggestion was made that behavioral inhibition might index components of asthma that are not immune-related. In the present study, we prospectively examined the relationship between behavioral inhibition and airway hyperresponsiveness, and whether hormonal and immune measures often associated with asthma were associated with behavioral inhibition and/or airway hyperresponsiveness. In a sample of 49 yearling rhesus monkeys (mean = 1.25 years, n = 24 behaviorally inhibited animals), we measured in vitro cytokine levels (IL-4, IL-10, IL-12, IFN-γ) in response to stimulation, as well as peripheral blood cell percentages, cortisol levels, and percentage of regulatory T-cells (CD3+CD4+CD25+FOXP3+). Airway reactivity was assessed using an inhaled methacholine challenge. Bronchoalveolar lavage was performed and the proportion of immune cells was determined. Behaviorally inhibited monkeys had airway hyperresponsiveness as indicated by the methacholine challenge (p = 0.031), confirming our earlier retrospective result. Airway hyperresponsiveness was also associated with lower lymphocyte percentages in lavage fluid and marginally lower plasma cortisol concentrations. However, none of the tested measures was significantly related to both behavioral inhibition and airway hyperresponsiveness, and so could not mediate their relationship. Airway hyperresponsiveness is common to atopic and non-atopic asthma and behavioral inhibition has been related to altered autonomic activity in other studies. Our results suggest that behavioral inhibition might index an autonomically mediated reactive airway phenotype, and that a variety of stimuli (including inflammation within lung tissue that is not specifically associated with behavioral inhibition) may trigger the airways response.
Current cigarette smoking is associated with reduced acute responses to corticosteroids and worse clinical outcomes in stable chronic asthma. The mechanism by which current smoking promotes this altered behavior is currently unclear. Whilst cytokines can induce corticosteroid insensitivity in-vitro, how current and former smoking affects airway cytokine concentrations and their responses to oral corticosteroids in stable chronic asthma is unclear.
To examine blood and sputum cytokine concentrations in never, ex and current smokers with asthma before and after oral corticosteroids.
Exploratory study utilizing two weeks of oral dexamethasone (equivalent to 40 mg/day prednisolone) in 22 current, 21 never and 10 ex-smokers with asthma. Induced sputum supernatant and plasma was obtained before and after oral dexamethasone. 25 cytokines were measured by multiplex microbead system (Invitrogen, UK) on a Luminex platform.
Smokers with asthma had elevated sputum cytokine interleukin (IL) -6, -7, and -12 concentrations compared to never smokers with asthma. Few sputum cytokine concentrations changed in response to dexamethasone IL-17 and IFNα increased in smokers, CCL4 increased in never smokers and CCL5 and CXCL10 reduced in ex-smokers with asthma. Ex-smokers with asthma appeared to have evidence of an ongoing corticosteroid resistant elevation of cytokines despite smoking cessation. Several plasma cytokines were lower in smokers with asthma compared to never smokers with asthma.
Cigarette smoking in asthma is associated with a corticosteroid insensitive increase in multiple airway cytokines. Distinct airway cytokine profiles are present in current smokers and never smokers with asthma and could provide an explanatory mechanism for the altered clinical behavior observed in smokers with asthma.
Major histocompatibility complex class I (MHC I) on antigen presenting cells (APCs) is a potent molecule to activate CD8+ T cells and initiate immunity. P2X7 receptors (P2X7Rs) are present on the plasma membrane of APCs to sense the extracellular danger signal adenosine-5′-triphosphate (ATP). P2X7R activates the inflammasome and the release of IL-1β in macrophages and other immune cells to initiate the inflammatory response. Here we show that P2X7R stimulation by ATP in APCs decreased the amount of MHC I at the plasma membrane. Specific antagonism or genetic ablation of P2X7R inhibited the effects of ATP on levels of cellular MHC I. Furthermore, P2X7R stimulation was able to inhibit activation of CD8+ T cells via specific MHC I-oligopeptide complexes. Our study suggests that P2X7R activation on APCs is a novel inhibitor of adaptive CD8+ T cell immunity.
HIF-1α is a transcription factor that is activated during hypoxia and inflammation and is a key regulator of angiogenesis in vivo. During the development of asthma, peribronchial angiogenesis is induced in response to aeroallergens and is thought to be an important feature of sustained chronic allergic inflammation. Recently, elevated HIF-1α levels have been demonstrated in both the lung tissue and bronchoalveolar lavage of allergic patients, respectively. Therefore, we investigated the role of HIF-1α on the development of angiogenesis and inflammation following acute and chronic allergen exposure. Our data shows that intranasal exposure to house dust mite (HDM) increases the expression of HIF-1α in the lung, whilst reducing the expression of the HIF-1α negative regulators, PHD1 and PHD3. Blockade of HIF-1α in vivo, significantly decreased allergic inflammation and eosinophilia induced by allergen, due to a reduction in the levels of IL-5 and Eotaxin-2. Importantly, HIF-1α blockade significantly decreased levels of VEGF-A and CXCL1 in the lungs, which in turn led to a profound decrease in the recruitment of endothelial progenitor cells and a reduction of peribronchial angiogenesis. Furthermore, HDM or IL-4 treatment of primary lung macrophages resulted in significant production of both VEGF-A and CXCL1; inhibition of HIF-1α activity abrogated the production of these factors via an up-regulation of PHD1 and PHD3. These findings suggest that novel strategies to reduce the expression and activation of HIF-1α in lung macrophages may be used to attenuate allergen-induced airway inflammation and angiogenesis through the modulation of VEGF-A and CXCL1 expression.
This study provides new insights into the role of HIF-1α in the development of peribronchial angiogenesis and inflammation in a murine model of allergic airway disease. These findings indicate that strategies to reduce activation of macrophage derived HIF-1α may be used as a target to improve asthma pathology.
Vitamin D may be essential for restricting the development and severity of allergic diseases and asthma, but a direct causal link between vitamin D deficiency and asthma has yet to be established. We have developed a ‘low dose’ model of allergic airway disease induced by intraperitoneal injection with ovalbumin (1 µg) and aluminium hydroxide (0.2 mg) in which characteristics of atopic asthma are recapitulated, including airway hyperresponsiveness, antigen-specific immunoglobulin type-E and lung inflammation. We assessed the effects of vitamin D deficiency throughout life (from conception until adulthood) on the severity of ovalbumin-induced allergic airway disease in vitamin D-replete and -deficient BALB/c mice using this model. Vitamin D had protective effects such that deficiency significantly enhanced eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male but not female mice. Vitamin D also suppressed the proliferation and T helper cell type-2 cytokine-secreting capacity of airway-draining lymph node cells from both male and female mice. Supplementation of initially vitamin D-deficient mice with vitamin D for four weeks returned serum 25-hydroxyvitamin D to levels observed in initially vitamin D-replete mice, and also suppressed eosinophil and neutrophil numbers in the bronchoalveolar lavage fluid of male mice. Using generic 16 S rRNA primers, increased bacterial levels were detected in the lungs of initially vitamin D-deficient male mice, which were also reduced by vitamin D supplementation. These results indicate that vitamin D controls granulocyte levels in the bronchoalveolar lavage fluid in an allergen-sensitive manner, and may contribute towards the severity of asthma in a gender-specific fashion through regulation of respiratory bacteria.
The Ca2+-activated K+ channel KCa3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of KCa3.1 modifies experimental asthma in sheep.
Methodology and Principal Findings
Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the KCa3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.
These findings suggest that KCa3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of KCa3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.
The expression of HDAC2 is reported as reduced in chronic obstructive pulmonary disease (COPD). We assessed HDAC2 expression within the airways of smokers and subjects with COPD and effects of inhaled corticosteroids (ICS), using immuno-histology to contrast with previous molecular methodology.
Endobronchial biopsies (ebb) from current smokers with COPD (COPD-CS; n = 15), ex-smokers with COPD (COPD-ES; n = 17), smokers with normal lung function (NS; n = 16) and normal controls (NC; n = 9) were immunostained for HDAC2. A double-blinded, randomized, placebo-controlled 6 months intervention study assessed effects of ICS on HDAC2 in 34 COPD subjects.
There was no difference in epithelial HDAC2 staining in all groups. There was a significant reduction in total cell numbers in the lamina propria (LP) in COPD-CS and NS (p<0.05). LP cellularity correlated inversely with smoking history in COPD-CS (R = −0.8, p<0.003). HDAC2 expression increased markedly in NS (p<0.001); in contrast COPD-CS was associated with suppressed signal (p<0.03), while normal in COPD-ES. ICS did not affect HDAC2 cell staining.
Our findings suggest that airway HDAC2 expression is increased in the LP by smoking itself, but is reduced in COPD. Ex-smokers have normalised HDAC2 cell expression, but ICS had no effect. The paper emphasise the pit-falls of relying on molecular data alone to define airway changes.
Clinical Trial Registration Information:
Name of registry
The Australian New Zealand Clinical Trials Registry (ANZCTR)
Cardiovascular disease (CVD) can be detected and quantified by analysis of the electrocardiogram (ECG); however the effects of smoking and smoking cessation on the ECG have not been characterized.
Standard 12-lead ECGs were performed at baseline and 3 years after subjects enrolled in a prospective, randomized, placebo-controlled clinical trial of smoking cessation pharmacotherapies. ECGs were interpreted using the Minnesota Code ECG Classification. The effects of (i) smoking burden on the prevalence of ECG findings at baseline, and (ii) smoking and smoking cessation on ECG changes after 3 years were investigated by multivariable and multinomial regression analyses.
At baseline, 532 smokers were (mean [SD]) 43.3 (11.5) years old, smoked 20.6 (7.9) cigarettes/day, with a smoking burden of 26.7 (18.6) pack-years. Major and minor ECG criteria were identified in 87 (16.4%) and 131 (24.6%) of subjects, respectively. After adjusting for demographic data and known CVD risk factors, higher pack-years was associated with major ECG abnormalities (p = 0.02), but current cigarettes/day (p = 0.23) was not. After 3 years, 42.9% of subjects were abstinent from smoking. New major and minor ECG criteria were observed in 7.2% and 15.6% of subjects respectively, but in similar numbers of abstinent subjects and continuing smokers (p>0.2 for both). Continuing smokers showed significant reduction in current smoking (–8.4 [8.8] cigarettes/day, p<0.001) compared to baseline.
In conclusion, major ECG abnormalities are independently associated with lifetime smoking burden. After 3 years, smoking cessation was not associated with a decrease in ECG abnormalities, although cigarettes smoked/day decreased among continuing smokers.
In experimental conditions alveolar fluid clearance is controlled by alveolar β2-adrenergic receptors. We hypothesized that if this occurs in humans, then non-selective β-blockers should reduce the membrane diffusing capacity (DM), an index of lung interstitial fluid homeostasis. Moreover, we wondered whether this effect is potentiated by saline solution infusion, an intervention expected to cause interstitial lung edema. Since fluid retention within the lungs might trigger excessive ventilation during exercise, we also hypothesized that after the β2-blockade ventilation increased in excess to CO2 output and this was further enhanced by interstitial edema.
Methods and Results
22 healthy males took part in the study. On day 1, spirometry, lung diffusion for carbon monoxide (DLCO) including its subcomponents DM and capillary volume (VCap), and cardiopulmonary exercise test were performed. On day 2, these tests were repeated after rapid 25 ml/kg saline infusion. Then, in random order 11 subjects were assigned to oral treatment with Carvedilol (CARV) and 11 to Bisoprolol (BISOPR). When heart rate fell at least by 10 beats·min−1, the tests were repeated before (day 3) and after saline infusion (day 4). CARV but not BISOPR, decreased DM (−13±7%, p = 0.001) and increased VCap (+20±22%, p = 0.016) and VE/VCO2 slope (+12±8%, p<0.01). These changes further increased after saline: −18±13% for DM (p<0.01), +44±28% for VCap (p<0.001), and +20±10% for VE/VCO2 slope (p<0.001).
These findings support the hypothesis that in humans in vivo the β2-alveolar receptors contribute to control alveolar fluid clearance and that interstitial lung fluid may trigger exercise hyperventilation.
Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10–dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.
Hypomorphic mutations in the bone morphogenic protein receptor (BMPR2) confer a much greater risk for developing pulmonary arterial hypertension (PAH). However, not all carriers of a mutation in the BMPR2 gene suffer from PAH. We have previously shown that prolonged T helper 2 (Th2) responses in the lungs to a mild antigen delivered via the airways induce severe pulmonary arterial remodeling, but no pulmonary hypertension. The current studies were designed to test the idea that Th2 responses to a mild antigen together with the expression of a hypomorphic BMPR2 gene would trigger pulmonary hypertension.
Mice that expressed a hypomorphic BMPR2 transgene (transgene-positive) and transgene-negative mice were either exposed to saline, or primed and exposed to a mild antigen (Ovalbumin) over a prolonged period of time. Only transgene-positive but not transgene-negative mice exposed to antigen developed significantly increased right ventricular systolic pressures, while both groups showed pulmonary artery remodeling with severe muscularization and airway inflammation to a similar degree. Antigen exposure resulted in a smaller increase in the percentage of Interleukin (IL)-13 positive T cells in the lymph nodes, and in a smaller increase in resistin-like-molecule (RELM)α expression and a decreased ratio of expression of IL-33 relative to its receptor (IL-1-receptor-like 1, IL1RL1-ST2) in the right ventricles of transgene-positive mice compared to transgene-negative animals. Furthermore, only antigen-challenged transgene-positive mice showed a significant increase in Interferon (IFN)γ positive T cells over saline-exposed controls.
Our study suggests that exposure with a mild Th2 antigen can trigger pulmonary hypertension on the background of the expression of a hypomorphic BMPR2 gene and that conversely, the expression of the hypomorphic BMPR2 gene can alter the immune response to a mild, inhaled antigen.
IL-18 plays a key role in the pathogenesis of pulmonary inflammatory diseases including pulmonary infection, pulmonary fibrosis, lung injury and chronic obstructive pulmonary disease (COPD). However, it is unknown whether IL-18 plays any role in the pathogenesis of asthma. We hypothesized that overexpression of mature IL-18 protein in the lungs may exacerbate disease activities of asthma. We established lung-specific IL-18 transgenic mice on a Balb/c genetic background. Female mice sensitized– and challenged– with antigen (ovalbumin) were used as a mouse asthma model. Pulmonary inflammation and emphysema were not observed in the lungs of naïve transgenic mice. However, airway hyperresponsiveness and airway inflammatory cells accompanied with CD4+ T cells, CD8+ T cells, eosinophils, neutrophils, and macrophages were significantly increased in ovalbumin-sensitized and challenged transgenic mice, as compared to wild type Balb/c mice. We also demonstrate that IL-18 induces IFN-γ, IL-13, and eotaxin in the lungs of ovalbumin-sensitized and challenged transgenic mice along with an increase in IL-13 producing CD4+ T cells. Treatment with anti-CD4 monoclonal antibody or deletion of the IL-13 gene improves ovalbumin-induced airway hyperresponsiveness and reduces airway inflammatory cells in transgenic mice. Overexpressing the IL-18 protein in the lungs induces type 1 and type 2 cytokines and airway inflammation, and results in increasing airway hyperresponsiveness via CD4+ T cells and IL-13 in asthma.
Despite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration.
We performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject’s response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms.
Among subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×104 per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens.
In mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.
The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored.
Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity.
Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is warranted and could potentially lead to the development of improved diagnostic and therapeutic tools.
Imaging studies have demonstrated that ventilation during bronchoconstriction in subjects with asthma is patchy with large ventilation defective areas (Vdefs). Based on a theoretical model, we postulated that during bronchoconstriction, as smooth muscle force activation increases, a patchy distribution of ventilation should emerge, even in the presence of minimal heterogeneity the lung. We therefore theorized that in normal lungs, Vdefs should also emerge in regions of the lung with reduced expansion.
We studied 12 healthy subjects to evaluate whether Vdefs formed during bronchoconstriction, and compared their Vdefs with those observed in 9 subjects with mild asthma.
Spirometry, low frequency (0.15 Hz) lung elastance and resistance, and regional ventilation by intravenous 13NN-saline positron emission tomography were measured before and after a challenge with nebulized methacholine. Vdefs were defined as regions with elevated residual 13NN after a period of washout. The average location, ventilation, volume, and fractional gas content of the Vdefs, relative to those of the rest of the lung, were calculated for both groups.
Consistent with the predictions of the theoretical model, both healthy subjects and those with asthma developed Vdefs. These Vdefs tended to form in regions that, at baseline, had a lower degree of lung inflation and, in healthy subjects, tended to occur in more dependent locations than in subjects with asthma.
The formation of Vdefs is determined by the state of inflation prior to bronchoconstriction.
Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.
Peanut allergy is the leading cause of deaths due to food-induced anaphylaxis but despite continued research, there are currently no specific treatments available. Challenge testing is limited in patients due to the high risk of adverse reactions, emphasising the need for an appropriate animal model. In the present study we examine the induction of allergic responses in a sheep model for peanut allergy. Sheep were sensitised with peanut (PN) extract and in separate injections with ovalbumin (OVA) or house dust mite (HDM) extract. Serum PN-specific IgE responses were detected in 40–50% of immunised sheep, while only 10% (1 of 10 sheep) showed detectable OVA-specific IgE. All PN-allergic sheep tested showed an Ara h 1-specific IgE response, while four out of five allergic sheep showed an Ara h 2-specific IgE response. Animals with high serum IgE levels to HDM were also PN IgE-positive. Of the PN-sensitised animals with high PN-specific IgE, 80% also showed an immediate hypersensitivity reaction following an intradermal PN injection. This new large animal model of peanut allergy may provide a useful tool for future investigations of allergen-associated immune mechanisms and specific immunotherapy.
Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c+CD14− and CD14+ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4+ or CD8+T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c+CD14−DDCs were able to differentiate naïve CD4+T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4+T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4+ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.
The contraction phase of antigen-specific immune responses involves the apoptotic loss of numerous activated lymphocytes. While apoptotic cells are known to induce immune suppression, the mechanisms involved therein are still ambiguous. Some reports have speculated that macrophages can induce regulatory T cells (Tregs) after engulfing apoptotic cells. In this study, we showed that dendritic cells (DCs) that phagocytose apoptotic T cells acquire inhibitory function (named DCapos) toward CD4+ and CD8+ T cells. These inhibitory DCs could not induce the generation of Tregs, but they were found to directly inhibit mDCs that initiate CD4+ and CD8+ T cell proliferation both in vitro and in vivo. Soluble factors including NO play a role in the DCapos-induced suppression of CD4+ and CD8+ T cell proliferation. Further results showed that STAT3 phosphorylation and inducible nitric oxide synthase (iNOS) generation were enhanced when DCs were co-cultured with apoptotic cells. Both iNOS transcription and NO secretion were inhibited in the presence of the specific p-STAT3 inhibitor JSI-124. All the data indicated that apoptotic cells could turn DCs to inhibitory DCs, which might play important roles in the suppression of immune responses. STAT3 activation and the consequent release of NO are responsible for the inhibitory functions of DCapos.
The cannabinoid system is known to be involved in the regulation of inflammatory processes. Therefore, drugs targeting cannabinoid receptors are considered as candidates for anti-inflammatory and tissue protective therapy. We demonstrated that the prototypical cannabinoid agonist R(+)WIN55,212-2 (WIN) reduced the secretion of matrix metalloproteinase-9 (MMP-9) in a murine model of cigarette-smoke induced lung inflammation. In experiments using primary cells and cell lines of the monocyte-macrophage-system we found that binding of the cannabinoid-receptor agonist WIN to a stereo-selective, specific binding site in cells of the monocyte-macrophage-system induced a significant down-regulation of MMP-9 secretion and disturbance of intracellular processing, which subsequently down-regulated MMP-9 mRNA expression via a ERK1/2-phosphorylation-dependent pathway. Surprisingly, the anti-inflammatory effect was independent from classical cannabinoid receptors. Our experiments supposed an involvement of TRPV1, but other yet unidentified sites are also possible. We conclude that cannabinoid-induced control of MMP-9 in the monocyte-macrophage system via a cannabinoid-receptor independent pathway represents a general option for tissue protection during inflammation, such as during lung inflammation and other diseases associated with inflammatory tissue damage.
Clinical interpretation of bronchoalveolar lavage fluid results is dependent on the availability of reference values for healthy individuals. Only a few studies have published such reference values and the applicability of results is restricted by small sample sizes and the limited representativeness of the study population. We aim to investigate the influence of age, gender, collection site and season on bronchoalveolar lavage fluid results and to establish reference values for use in clinical practice.
Bronchoalveolar lavage fluid data from 295 healthy never-smoking volunteers, investigated during 1990–2009, were analyzed retrospectively. 47 volunteers had 2–5 repeat lavages during the course of several years. Fluid recovery, total number of cells, cell concentration, and differential cell counts on cytospin prepared slides were recorded. Reference values, as represented by the 5th to the 95th percentile, were 72–96% for macrophages, 2–26% for lymphocytes, 0–4% for neutrophils and 0–1% for eosinophils. Basophils and mast cells were rare. When repeat lavages were performed, there was a relatively large intra-individual variability, mainly for macrophages and lymphocytes. An age dependent decrease of lavage fluid return was present, but there was no age dependent correlation with any of the other BALF parameters. The BALF cell parameters were independent of gender, season and site (lingula vs. middle lobe).
Our data show that bronchoalveolar lavage fluid cell differential count is independent of age, gender, season and collection site (RML or lingua). It therefore seems acceptable to use the same reference values for all never-smoking individuals.